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With cell cultures in the lab - a day with doctoral candidates in biological research

BY Anke Wilde

When we promise to be quiet as mice so as not to disturb the three researchers, MinChi and Frank exchange amused glances. Mice tend to play a very special role in biological research - as test subjects. Yet the three doctoral candidates are willing to allow us to take a closer look over their shoulders. It makes one particular thing clear: a great deal of patience and staying power are needed in the lab.

With cell cultures in the lab - a day with doctoral candidates in biological researchNeurobiologist Petya Georgieva is doing her doctorate at the Max Delbrück Centre on the subject of microglia cells
It is like a lab out of a chemistry teacher's dream: white tiled workspaces, shelves lined with countless test tubes, bulbs and bottles, not a sound other than a radio in the background. Petya Georgieva has hardly pulled on clean latex gloves and arranged her samples and equipment around her and she is already lost entirely in a world of her own. The work processes of the native Bulgarian always follow the same procedure: fill a pipette with the desired amount of liquid using a little wheel, fix on a new tip, draw up the substance and eject it into a test tube. Then with a click of the pipette, the tip drops into a tabletop waste bin, and the same procedure is repeated.

Petya is a neurobiologist and is writing her doctoral thesis as a member of Professor Dr. Helmut Kettenmann's work group at the Max Delbrück Centre for Molecular Medicine in Berlin. The work group focuses on one very special type of cell: the microglia cells. In healthy brains, they ensure that all the other cells function correctly. Proteins on their surface tell them if a cell goes astray and its biochemical environment changes, for example. The microglia cells then trigger their natural cell death. However, tumour cells halt this protection mechanism of the brain. And not only that: the tumour cells reverse the polarity of the microglia cells making them support the malignant cells in their unabated proliferation. A cure does not yet exist for this disease, and in most cases, those affected often only have just a few months to live following diagnosis.

The long journey before the experiment

Petya is interested in the fundamental characteristics of the microglia cells in both diseased and healthy brains. Microglia cells have different states of activation, she explains. The differences that exist and how the cells move from one state to another forms the topic of her doctoral thesis. She originally wanted to consider another aspect, however her experiments did not lead to the anticipated results and she had to select another topic. Sometimes you simply have to accept that you cannot get any further, the 27-year-old affirms.

Most work processes in biological research are purely routine. Weeks must often be spent preparing the experiments that will potentially lead to new findings. Today Petya will investigate how many cells each of her cell samples actually contains. This can be determined bx examining the concentration of proteins. A ready-made assay kit is available to establish this - a complete package containing the chemicals required for each experiment. Kits such as this one exist for many of the tests performed in everyday molecular biology - indispensable and time-saving aids manufactured by companies specialising in these test systems.

Comfortable conditions for the cells

A few labs away, MinChi Ku is checking her cell cultures. Similar to Petya, the Taiwanese student is doing her doctorate under the supervision of Helmut Kettenmann, though she already completed her doctoral thesis two months ago. Her topic: the interaction between microglia cells and tumour cells. During this, she discovered a special protein, which is actually required for the growth of nerve cells. MinChi learned in her experiments that this protein is also released by the tumour cells and gives the microglia cells the signal that they should stimulate growth of the tumour cells. MinChi will soon begin a postdoctorate degree here at the MDC, and wishes to pursue several side-projects in the meantime. There's no end to the work of a researcher, says the 34-year-old.

Her cell cultures are stored in a special incubator that looks like a refrigerator but in which the ideal conditions of 37 degrees centigrade and 80 per cent humidity are constantly maintained. The cells are like babies, MinChi says. They should feel comfortable and be allowed to grow, and, therefore, cannot remain outside for a long time. Still invisible to the beholder, they swim in a pink nutrient solution in small sealed plastic Petri dishes. The nutrient solutions with which the cell cultures are regularly fed are stored in fridges and warmed to 37 degrees centigrade before being fed to the cells to ensure that the cells do not suffer cold shock.

MinChi takes a look at her cell culture under the microscope to ensure they really are ok. She has different cell types: rather triangular microglia cells from mice and round tumour cells cultivated for research purposes from a human cell line, both of which are handled differently. The cells look accordingly different: some are larger, some are smaller - and this despite belonging to the same type. And some haven't survived the treatment administered by MinChi.

She will feed the cells special proteins later on and observe whether her cells move towards them or not. This takes place at workstations that are separated from the rest of the lab by a glass pane. Around just 20 centimetres at the bottom remain open for the scientists to reach in and work behind the pane - with gloved hands, of course, disinfected with alcohol beforehand. Slightly higher air pressure prevails within the glass box and clean air is constantly blown at the opening by a fan - simple protection against dust and germs in the ambient air, which would contaminate the cell cultures within a matter of seconds otherwise.

Designer molecular chains

Meanwhile, Frank Szulzewsky checks a DNA sequence on his computer, which he must modify for an experiment. The 27-year-old biotechnologist joined Helmut Kettenmann's work group just one year ago and, similar to MinChi, is doing his doctorate on the interaction between microglia cells and tumour cells. In his work, the focus is on the extent to which the tumour jumbles the genetic programming of the microglia cells, and how this can be prevented.

Frank thoroughly enjoys designing molecules on the computer to suit exactly how he needs them to be. The DNA sequence on his screen does not simply end up as an elaborate diagram. It is merely a seemingly random combination of just four continually recurring letters over several lines: A, C, G, T, the building blocks of DNA. The molecular chains contain a segment of the blueprint of a cell, which ensures that it glows green. Frank will have to cut off the rest of the molecular chain though, as building instructions for the cells that he does not need are located there. So-called restriction enzymes, which work on one section of the DNA with a specific chemical structure, help him with the cutting. The biotechnologist can locate these places near the greening sequence with his computer program.

Steps en route to an experiment

He wants to order the restriction enzymes from a supplier today, and will then receive it as a solution in a little vial next week. He will mix this with the liquid in which his DNA molecules are swimming. At the end of the reaction, he will obtain the fine, neatly cut greening snippets. This also forms part of a biotechnologist's routine - prompting reactions between different substances that cannot be seen and during which one must place a great deal of trust in the expertise of others.

He wants to implant the cut sequence into bacteria next week. The bacteria reproduce and cost-efficiently proliferate the molecular chains that make the cells glow green. Frank will then introduce these into viruses, which will infect his cell cultures (passing on this green molecule). If all goes the plan, the cells will do exactly what he wants them to do in the end: glow green - meaning that they can be identified more easily under the microscope in more complex cell cultures. All in all, a genetic engineering project lasting several weeks.

The colleagues in the lab play an important role within this: they provide support, which makes it easier to overcome any failures you might experience. They have all frequently experienced the lows that must be dealt with when an experiment refuses to work or the course followed turns out to be a dead end. Expert advice or simply colleagues' sympathy is worth a great deal here.

The point at which everything becomes clear

MinChi is currently pursuing one of her favourite pastimes - imaging processes that take place in tissue working with a so-called confocal microscope - a machine that easily costs something in the realms of a six-digit figure - she is enlarging and scanning a cancerous area of a mouse's brain. The colourful images produced illustrate the tremendous amount of progress that has been made in biotechnology in the space of just a few decades.

The mice MinChi uses for her tests are genetically modified. Special markers such as those Frank previously showed on the computer have been added to their genetic code, which causes each cell type in the brain to assume a different colour. Specific proteins produced by the cells can also be stained. The microscope connected to a computer can differentiate between the colour spectrums. Thus with each individual scan, one specific cell type or protein is pictured from one frame, stained in violet, sky blue or lurid green. Superimposing all scans on one another creates a kind of family portrait revealing all the relationships and hostilities between each person - or rather cell : how individuals engage with one another, which powers are in play, who is excluded, and who seeks to break down existing structures. Or, in the words of neuroscientist MinChi, what the relationships between the cells are, and which cells and cell types produce which protein in the tumour tissue. At any rate, this is the point at which new scientific findings are possible.

Mice and men

Before our departure, Petya has one more thing on her mind: mice. At the MDC, a new animal facility is currently being built, and this has attracted the attention of many animal rights activists. However mice are absolutely essential in research, Petya stresses. Animal trials are only performed under the proviso that the trials in the cell culture were successfully completed. Conditions are also extremely stringent and each individual test must be justified for each project and approved by the responsible authorities. Prior to this, they are not ethically justifiable and would not be performed. Those wishing to do without animal trials would have no other choice but to test active substances on humans straight away and that would be far worse. The subject is an extremely serious one for Petya, which is why it is simply the price one must pay should one wish to find effective means to combat cancer and other terrible diseases.

academics :: December 2012